Fluorescence is a photochemical phenomenon during which a photon within a specific range of light wavelengths (excitation wavelengths) is absorbed by an indicator molecule, thereby exciting an electron to a higher energy state. When the excited electron decays back to its original ground state, the absorbed energy is released either radiatively as a second photon of light at a longer wavelength (emission wavelengths), or dissipated non-radiatively into the environment around the indicator molecule. Fluorescence is the release of this second, longer wavelength photon from the indicator molecule. The total time between absorption of the excitation photon and the fluorescence emission is typically on the order of 10.sup.-8 s for transitions involving visible light.
The phenomenon of fluorescence has been applied for many years to the field of chemical detection and identification of species. Many fluorescence-based chemical sensors indirectly detect the presence of an analyte using fluorescent compounds whose fluorescence properties change in response to changes in the concentration of the analyte. Alternatively, the competitive binding of the analyte to a receptor molecule versus binding by a fluorescent labeled analog of the analyte can be detected. Much effort has been expended developing sensors to detect the light response of fluorescing materials because the fluorescent light response may be vanishingly small and therefore difficult to detect.
A desirable format for fluorescence sensing is involves immobilizing an analyte-sensitive fluorescent compound to the surface of a waveguide and then introducing the treated surface into a solution containing a target analyte. Light having wavelengths known to excite the fluorescence of the immobilized compound is passed into the waveguide. The electromagnetic wave generated at the surface of the waveguide, known as the evanescent field, excites the very thin layer of the immobilized compound. The fluorescence response of the compound is then collected by the same waveguide and measured at a point distal to the excited layer.
Such optical interaction then permits one to assay a variety of chemical and biological materials. A number of such systems using internal total reflection spectroscopy for an assay are known and have been described, for example, in U.S. Pat. No. 4,133,639 which discloses a system that measures fluorescence induced by the optical interaction; in U.S. Pat. No. 4,050,895 which describes a system based on absorption of the evanescent wave by the analyte; in U.S. Pat. Nos. 5,738,992, and 5,525,466 which describes a system based on absorption of the evanescent wave by an indicator species immobilized at the surface of a waveguide; and in U.S. Pat. No. 4,447,546 which describes a fluorescence immunoassay system.
A popular geometry for such evanescent field sensors is the surface of the core of a fiber optic waveguide. Excitation light is collected and delivered to a distal portion of the fiber where the cladding has been removed to expose the surface of the core and on which a covalently bonded indicator species has been immobilized. Light introduced into the proximal end of the waveguide is totally internally reflected in the optically dense medium of the waveguide, and generates the evanescent wave at the surface of the exposed waveguide, which extends only a fraction of a wavelength into the test solution. This penetration, however, is sufficient to permit substantial optical interaction between the evanescent wave component and the immobilized indicator species with which the analyte in the test solution interacts. A small percentage of the emitted fluorescent light is coupled back into the trapped mode of the waveguide and measured at a proximal end of the fiber. Although this geometry offers many advantages such as small size and remote sensing capability, the collection of only a small part of the total excited fluorescent light limits the sensitivity and cost of this sensor design.
Although the use of a fine glass fiber as a waveguide offers several advantages such as small size and remote sensing, the amount of total excited light limits the sensitivity and cost of these sensors. To increase the efficiency of fluorescence collection, several approaches have been proposed. U.S. Pat. No. 4,654,532 which discloses a method for improving the numerical aperture of a fiber optic waveguide; and U.S. Pat. No. 5,138,153 discloses a fiber optic waveguide having a membrane coating. Another possible technique for increasing the percentage of collected fluorescence is the application of a thin metal film onto the surface of the core of an optical fiber. It has been shown that the fluorescence emitted beyond the supercritical angle at a planar metal-film-coated dielectric interface can be approximately 2-3 times greater than that for a bare dielectric surface. However, fluorescence excited close to the surface of the metal film is quenched, limiting the total amount of collectable fluorescence. What is needed, therefore, is a method for increasing the excitation and collection efficiency of fluorescence emission in a fiber optic waveguide which is both simple and inexpensive.
One possible method for increasing excited and collected fluorescence at a totally internally reflecting surface might be the application of a thin film having a high refractive index, n.sub.r, to the surface of the fiber optic. Such a high n.sub.r film is postulated to increase the excitation and collection of fluorescence through the greater depth of penetration of the evanescent field and the enhancement of the evanescent field intensity.